Compositions and Methods for Regulating Intramembrane Proteases

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Princeton University Invention # 07-2348


Intramembrane proteolysis is a widely conserved regulatory mechanism in species ranging from bacteria to humans. The first description of intramembrane proteolysis came from the investigation of cholesterol homeostasis, where the ER membrane-bound transcriptional factor SREBP must be cleaved by an integral-membrane protease, known as site-2 protease (S2P). This cleavage results in the release of the N-terminal domain of SREBP, which contains a DNA-binding domain and a transactivation domain. The N-terminal domain of SREBP regulates transcription of a number of genes that collectively control biosynthesis of cholesterol and fatty acid. Another prominent example of intramembrane proteolysis is the proteolytic processing of the amyloid precursor protein (APP) by the intramembrane protease g-secretase, which is central to the development of Alzheimer¿s disease. The cleavage product of APP, amyloid b-peptide, exhibits pronounced toxicity to neuronal cells and is thought to contribute to Alzheimer¿s disease. More recently, study of epidermal growth factor receptor (EGFR) signaling in Drosophila identified rhomboid as an essential component in the signal-sending cells. Rhomboid, a putative intramembrane protease, cleaves the ligand Spitz, which is inactive in its full-length form, thus regulating EGFR signaling spatially and temporally.


There are four families of integral membrane proteins that are though to catalyze intramembrane proteolysis: the serine protease rhomboid, metalloprotease S2P, aspartyl proteases presenilin (catalytic subunit of g-secretase) and signal-peptide peptidase. The putative catalytic residues are predicted to be below the membrane surface and within the hydrophobic core of the proteases. In this case, since scission of peptide bonds requires the presence of water molecules, how do hydrophilic water molecules enter the active site? More importantly, if the active site is within the hydrophobic core of the proteases, how do the substrate proteins gain excess to the catalytic residues? Furthermore, are there some common principles that govern all four families of intramembrane proteases? These fundamental questions need to be addressed by a series of structures of the proteases at different stages of their action.


Researchers at Princeton University have determined the crystal structure of the transmembrane core domain of GlpG, a Rhomboid family intramembrane serine protease from Escherichia coli. The protein contains six transmembrane helices, with the catalytic Ser201 located at the amino-terminus of helix a4 approximately 10 Å below the membrane surface. Access to water molecules is provided by a central cavity that opens to the extracellular region and converges on Ser201. One of the two GlpG molecules in the asymmetric unit exhibits an open conformation at the active site, with the transmembrane helix a5 bent away from the rest of the molecule. Rigorous structural analysis was completed which indicates that substrate entry to the active site is most likely gated by the movement of the transmembrane helix a5 (TMH5). This conclusion may generally apply to all members of the rhomboid family intramembrane proteases as well as the other three families of intramembrane proteases, in which gating of substrate entry is proposed to be provided by a transmembrane helix.


This invention elucidates the method in which  the movement of TMH5 of a rhomboid intramembrane protease or the corresponding transmembrane helix in other families of intramembrane proteases is restrained by compounds and inhibitors. The restrained movement of TMH will result in a reduction of the intramembrane protease activity of intramembrane proteases. Reduced protease activity of intramembrane proteases provides a therapeutic treatment for diseases in which intramembrane proteases play a contributing role.  Such diseases could include neurological disorders, cardiovascular diseases, cancer, and others.




Wu, Z., Yan, W., Feng, L., Oberstein, A., Yan, H.,Baker, R., Gu, L., Jeffrey, P.,Urban, S., Shi, Y., Structural Analysis of a Rhomboid Family Intramembrane Protease Reveals a Gating Mechanisms for Substrate Entry, Nature, Strucutral Biology, Vol. 13, #12, December 2006.


Baker,R.P>, Young,K., Feng,L., Shi,Y., Urban,S., Enzymatic Analysis of a Rhomboid Intramembrane Protease Implicates Transmembrane Helix 5 as the Lateral Substrate Gate, PNAS USA, 2007, May 15:104(20) 8257-62.



Princeton is currently seeking industrial collaboration to commercialize this technology. Patent protection is pending.



For more information on Princeton University invention # 07-2348 please contact:


                        Laurie Tzodikov

                        Office of Technology Licensing and Intellectual Property

                        Princeton University

                        4 New South Building

                        Princeton, NJ 08544-0036

                        (609) 258-7256

                        (609) 258-1159 fax


Patent Information:
For Information, Contact:
Laurie Tzodikov
Licensing Associates
Princeton University
Yigong Shi
Nieng Yan